PROJECT SUMMARY/ ABSTRACT Three lessons of the PrEP (pre-exposure prophylaxis) clinical trials - that adherence is critical to effectiveness, that pharmacologic adherence measures are more reliable than self-report, and that daily pill-taking is difficult ?must be applied to the next phase of HIV prevention research. This phase will investigate oral PrEP roll-out and optimization in high incidence settings, as well as novel long-acting methods for preventing HIV infection, such as injectables or vaginal rings. The vaginal ring prevented HIV acquisition in two recent trials, but poor adherence to consistent ring insertion dampened overall effectiveness. Injectable PrEP with long-acting cabotegravir is of interest, but will require adequate drug levels to be effective, especially at the end of dosing intervals and with missed visits. Pharmacologic metrics integrate biology (pharmacokinetics, PK) and behavior (adherence) and will be crucial to interpreting effectiveness with real-world oral PrEP, rings and injectables. Our group has helped pioneer the use of small hair samples (which are easy to collect, store and ship) to monitor exposure (PK) and adherence to antiretrovirals (ARVs). During the first funding period of this R01, we made significant progress on our original aims, demonstrating that hair levels of ARVs, which monitor long-term exposure, are stronger predictors of treatment success than self-reported adherence (or plasma levels) in HIV-infected pregnant women, children, and adults. We have also shown preliminary utility of hair levels of tenofovir (TFV)/emtricitabine (FTC) to monitor adherence and toxicities with oral PrEP. This proposal will leverage three important trials to explore key knowledge gaps that will arise in the next phase of HIV prevention work: The Sustainable East Africa Research in Community Health (SEARCH) trial (Dr. Havlir, chair and co-I) has just launched a large study providing oral PrEP to at-risk individuals in 16 communities in Africa. The HIV Open-Label Prevention Extension (HOPE) study in the Microbicide Trials Network (MTN) (Dr. Baeten, chair and co-I) will assess open-label use of the dapivirine vaginal ring. The Long-Acting Antiretroviral Therapy in Non-adherent HIV-Infected Individuals Trial (A5359, Dr. Castillo- Mancilla, co-chair and co-I)) in the AIDS Clinical Trials Group (ACTG) will examine long-acting injectables in HIV-infected patients with a history of poor adherence. All 3 trials will collect hair and plasma for drug levels, and track robust outcomes, to allow us to 1) investigate hair levels as metrics of adherence with the vaginal ring and oral PrEP in Africa and examine patterns of adherence (e.g. daily, around periods of risk, just prior to visits) by combining data in plasma and hair; and 2) examine hair levels as easy-to-collect metrics for PK monitoring with the use of injectables. The overarching goal of this renewal is to develop an integrated package of highly predictive biologic adherence and pharmacokinetic measures spanning PrEP delivery methods and optimization strategies. Defining predictors and patterns of adherence to oral PrEP and rings, as well as metrics to monitor PK with injectables, will inform public health interventions in HIV prevention.